R2 Protein Research Articles

Assessing the yield and nutrient potential of horse gram mutants (Macrotyloma uniflorum Lam. Verdc.) an underutilized legume through a multi-environment-based experiment

The agronomic stability and nutritional importance of 30 (Test genotypes: 29 + Check: 1 = 30) promising horse gram mutants were evaluated in this multi-environment-based experiment (MEE). Attempts were made to (i) identify stable mutants for agronomic traits through AMMI and GGE biplot models, (ii) quantify nutritional traits, (iii) understand the linkage between yield and nutritional traits, and (iv) estimate physical (PP) and cooking properties (CP) of selected genotypes to fix their food-chain usability. The ANOVA of the pooled data exhibited significant differences among environments (E), genotypes (G), and GxE interaction. The combined AMMI and GGE results helped to identify a few good-yielding and stable genotypes (GYSM) (G1, G25, G3, and G27). The yield advantages of these GYSMs over the parent PAIYUR 2 are 42.99%, 34.63%, 28.68%, and 30.59% respectively. The nutrient profiling of mutants revealed (i) a significant coefficient of variation for macronutrients (fat: 29.98%; fibre: 20.72%, and protein: 5.01%), (ii) a good range of variation for micronutrients, and (iii) helped to identify macro (MaNSM) and micro nutrient-specific mutants (MiNSM). The relationship analysis between yield and nutrient traits ascertained that yield had (i) positivity with protein (r2 = 0.69) and negativity for micronutrients except for Mn (r2 = 0.63), Cu (r2 = 0.46), and B (r2 = 0.01) in GYSM, (ii) positivity with protein and fibre in MaNSM, and (iii) negativity with micronutrients in MiNSM. Of the GYSM, G1 and G25 offer scope for commercial exploitation, and their PP and CP analyses revealed that G1 can be used for pastry and baked product preparation while G25 for weaning foods. Cooking time exhibited positivity with seed size parameters and negativity with water absorption capacity (r2 = − 0.53). An LC–MS–MS-based amino acid (AA) fractionation study showed the effect of induced mutagenesis on the contents of amino acids and also revealed the significance of horse gram for its lysine and methionine contents.

Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, precise RNA-mediated insertion of transgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

Dietary intakes and nutritional status in adults with Muscular Dystrophy

Muscular dystrophy (MD) is a group of inherited conditions causing progressive muscle weakness and wasting, for which no widely implementable treatment currently exists, although resistance training (Bostock et al., 2019, Frontiers in Neurology, 10) and corticosteroid treatment (Angelini, 2007, Muscle & Nerve, 36, 424-435), have been shown to improve health-related quality of life (HQoL). Compared to physical activity, dietary manipulation may be more modifiable to improve dystrophic populations HQoL as disease outcomes often restrict physical capabilities. Data exploring the relationship between diet, disease and HQoL outcomes in adults with MD is insufficient. This study evaluated the habitual dietary intake of a sex-inclusive dystrophic population, to identify possible links between dietary intake, HQoL, and physical function. Participants included 5 women (age 65 ± 14 yrs, BMI 23.2 ± 2.7 kg/m2) and 10 men (age 55 ± 19 yrs, 31.6 ± 7.9 kg/m2) with different MDs (varied distribution and severity of muscle weakness). Participants' dietary intake data was collected using 3-day food records: analysed using Nutritics. Dominant hand maximal grip strength was measured using a hand-held dynamometer; upper extremity function and pain were assessed using the upper extremity functional index-15 and visual analogue scale, respectively. QoL was assessed through three tools: SF-36V2, Barthel Index and EQ5D, previously used in dystrophic populations. We observed that 50% of men and 20% of women did not meet the recommended daily intake (RDI) for protein (0.8 g·kg−1), however women (1.13 ± 0.26 g·kg−1) had increased protein intake compared to men (0.82 ± 0.27 g·kg−1, p < 0.05). Of all participants 40% had a daily fat intake exceeding the RDI, with women having significantly higher relative average fat intakes (women = 1.03 ± 0.36 g·kg−1, men = 0.68 ± 0.29 g·kg−1, p < 0.05). RDI for calcium was met by 46% participants (1000 mg) whilst all failed to meet the vitamin D RDI (10 μg). Positive correlations between age and protein intake as a percentage of RDI (r2 = 0.82), protein (r2 = 0.82), carbohydrate intake (r2 = 0.55) relative to body mass, vitamin D intake (r2 = 0.54), and total caloric intake (r2 = 0.59, all p< 0.05) were observed. No other significant associations between dietary habits, physical function or HQoL outcomes were apparent. Despite these findings being observed in a small population it is apparent that many adults with muscular dystrophy may fail to meet RDI for protein, vitamin D, and calcium. This places this population at risk for nutritional deficiencies, incurring negative impacts on the musculoskeletal system. Further study of nutritional status in a larger population sample is needed to confirm these findings.

Distinct and overlapping RNA determinants for binding and target-primed reverse transcription by Bombyx mori R2 retrotransposon protein.

Eukaryotic retrotransposons encode a reverse transcriptase that binds RNA to template DNA synthesis. The ancestral non-long terminal repeat (non-LTR) retrotransposons encode a protein that performs target-primed reverse transcription (TPRT), in which the nicked genomic target site initiates complementary DNA (cDNA)synthesis directly into the genome. The best understood model system for biochemical studies of TPRT is the R2 protein from the silk moth Bombyx mori. The R2 protein selectively binds the 3' untranslated region of its encoding RNA as template for DNA insertion to its target site in 28S ribosomal DNA. Here, binding and TPRT assays define RNA contributions to RNA-protein interaction, template use for TPRT and the fidelity of template positioning for TPRT cDNA synthesis. We quantify both sequence and structure contributions to protein-RNA interaction. RNA determinants of binding affinity overlap but are not equivalent to RNA features required for TPRT and its fidelity of template positioning for full-length TPRT cDNA synthesis. Additionally, we show that a previously implicated RNA-binding protein surface of R2 protein makes RNA binding affinity dependent on the presence of two stem-loops. Our findings inform evolutionary relationships across R2 retrotransposon RNAs and are a step toward understanding the mechanism and template specificity of non-LTR retrotransposon mobility.

Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci.

Current approaches for inserting autonomous transgenes into the genome, such as CRISPR-Cas9 or virus-based strategies, have limitations including low efficiency and high risk of untargeted genome mutagenesis. Here, we describe precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro transcribed RNAs: messenger RNA encoding avian R2 retroelement-protein and template RNA encoding a transgene of length validated up to 4 kb. The R2 protein coordinately recognizes the target site, nicks one strand at a precise location and primes complementary DNA synthesis for stable transgene insertion. With a cultured human primary cell line, over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. PRINT advantages include no extragenomic DNA, limiting risk of deleterious mutagenesis and innate immune responses, and the relatively low cost, rapid production and scalability of RNA-only delivery.

Decrease in Leptin Expression in the Liver after Prolonged Every-Other-Day Feeding in C57Bl/6 Male Mice

Background: The positive effects of prolonged every-other-day (EOD) feeding include decreased body weight and prolonged life span, but also changes in liver metabolism and functions. In the present paper, our aim was to examine the expression of adiponectin (ADIPOQ), leptin, and their receptors (ADIPOR1, ADIPOR2, and LEPR) in the liver tissue of EOD-fed mice in comparison to ad libitum (AL)-treated mice. Methods: After 9 months of EOD treatment, liver tissue was harvested and prepared for analysis. RT-PCR, protein semi-quantitative estimation, and cellular immunolocalization was performed. Results: We noted a decreased expression of leptin in the liver tissue of the EOD male mice in comparison to the AL mice on the protein level. ADIPOQ receptor R1 protein expression was decreased in the liver of EOD-fed male mice, while the expression of ADIPOR2 on the protein level was increased in the EOD animals. Conclusions: To the best of our knowledge, this is the first report showing ADIPOQ and leptin immunolocalization in the liver tissue in a dietary regime experiment. Decreases in leptin expression and IL-6 expression in liver tissue and increases in ADIPOR2 expression may be partly responsible for the beneficial effects of EOD treatment in the liver, including the decrease in inflammation. Further studies are needed to establish whether these changes depend on factors like the type of treatment, species, strain, gender, time of treatment, and others.

Structure of a ribonucleotide reductase R2 protein radical.

Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O-O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.

Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon

Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.

Well-Defined pH-Responsive Self-Assembled Block Copolymers for the Effective Codelivery of Doxorubicin and Antisense Oligonucleotide to Breast Cancer Cells.

The worldwide steady increase in the number of cancer patients motivates the development of innovative drug delivery systems for combination therapy as an effective clinical modality for cancer treatment. Here, we explored a design concept based on poly(ethylene glycol)-b-poly(2-(dimethylamino)ethyl methacrylate)-b-poly(2-hydroxyethyl methacrylate-formylbenzoic acid) [PEG-b-PDMAEMA-b-P(HEMA-FBA)] for the dual delivery of doxorubicin (DOX) and GTI2040 (an antisense oligonucleotide for ribonucleotide reductase inhibition) to MCF-7 breast cancer cells. PEG-b-PDMAEMA-b-PHEMA, the precursor copolymer, was prepared through chain extensions from a PEG-based macroinitiator via two consecutive atom transfer radical polymerization (ATRP) steps. Then, it was modified at the PHEMA block with 4-formylbenzoic acid (FBA) to install reactive aldehyde moieties. A pH-responsive polymer-drug conjugate (PDC) was obtained by conjugating DOX to the polymer structure via acid-labile imine linkages, and subsequently self-assembled in an aqueous solution to form DOX-loaded self-assembled nanoparticles (DOX-SAN) with a positively charged shell. DOX-SAN condensed readily with negatively charged GTI2040 to form GTI2040/DOX-SAN nanocomplexes. Gel-retardation assay confirmed the affinity between GTI2040 and DOX-SAN. The GTI2040/DOX-SAN nanocomplex at N/P ratio of 30 exhibited a volume-average hydrodynamic size of 136.4 nm and a zeta potential of 21.0 mV. The pH-sensitivity of DOX-SAN was confirmed by the DOX release study based on the significant cumulative DOX release at pH 5.5 relative to pH 7.4. Cellular uptake study demonstrated favorable accumulation of GTI2040/DOX-SAN inside MCF-7 cells compared with free GTI2040/DOX. In vitro cytotoxicity study indicated higher therapeutic efficacy of GTI2040/DOX-SAN relative to DOX-SAN alone because of the downregulation of the R2 protein of ribonucleotide reductase. These outcomes suggest that the self-assembled pH-responsive triblock copolymer is a promising platform for combination therapy, which may be more effective in combating cancer than individual therapies.

Estimating Crop Seed Composition Using Machine Learning from Multisensory UAV Data

The pre-harvest estimation of seed composition from standing crops is imperative for field management practices and plant phenotyping. This paper presents for the first time the potential of Unmanned Aerial Vehicles (UAV)-based high-resolution hyperspectral and LiDAR data acquired from in-season stand crops for estimating seed protein and oil compositions of soybean and corn using multisensory data fusion and automated machine learning. UAV-based hyperspectral and LiDAR data was collected during the growing season (reproductive stage five (R5)) of 2020 over a soybean test site near Columbia, Missouri and a cornfield at Urbana, Illinois, USA. Canopy spectral and texture features were extracted from hyperspectral imagery, and canopy structure features were derived from LiDAR point clouds. The extracted features were then used as input variables for automated machine-learning methods available with the H2O Automated Machine-Learning framework (H2O-AutoML). The results presented that: (1) UAV hyperspectral imagery can successfully predict both the protein and oil of soybean and corn with moderate accuracies; (2) canopy structure features derived from LiDAR point clouds yielded slightly poorer estimates of crop-seed composition compared to the hyperspectral data; (3) regardless of machine-learning methods, the combination of hyperspectral and LiDAR data outperformed the predictions using a single sensor alone, with an R2 of 0.79 and 0.67 for corn protein and oil and R2 of 0.64 and 0.56 for soybean protein and oil; and (4) the H2O-AutoML framework was found to be an efficient strategy for machine-learning-based data-driven model building. Among the specific regression methods evaluated in this study, the Gradient Boosting Machine (GBM) and Deep Neural Network (NN) exhibited superior performance to other methods. This study reveals opportunities and limitations for multisensory UAV data fusion and automated machine learning in estimating crop-seed composition.

Separate Effects of Foliar Applied Selenate and Zinc Oxide on the Accumulation of Macrominerals, Macronutrients and Bioactive Compounds in Two Pea (Pisum sativum L.) Seed Varieties.

Selenium (Se) and zinc (Zn) are important cofactors for antioxidant enzymes. Foliar Se/Zn application is a highly efficient strategy of plant biofortification. However, its effects on the accumulation of macrominerals, macronutrients and bioactive compounds in the pea plant (Pisum sativum L.) have been poorly investigated. A two-year pot experiment was performed to study responses of two pea varieties (Ambassador, Premium) to foliar-applied sodium selenate (0/50/100 g Se/ha) and zinc oxide (0/375/750 g Zn/ha) at the flowering stage. Concentrations of Ca, Mg, K, Na, soluble solids (SSC), protein, chlorophyll a and b, total chlorophyll, total carotenoids and total condensed tannins (TCT) were determined in seeds. Mg concentration in Ambassador and chlorophyll a concentration in Premium were positively affected, in part, by selenate and zinc oxide, respectively. Selenate and zinc oxide increased, in part, protein concentration in Premium. Highest protein concentration was found in Premium treated with 375 g Zn/ha (27.6% DW) vs. the control (26.6% DW). Significant (all p < 0.001) positive correlations were found, among others, between concentrations of Zn and Mg (r2 = 0.735) and between Zn and protein (r2 = 0.437) for Ambassador, and between Mg and protein (r2 = 0.682), between Zn and Mg (r2 = 0.807), as well as between Zn and protein (r2 = 0.884) for Premium. TCT significantly (all p < 0.05) and positively correlated with SSC (r2 = 0.131), chlorophyll b (r2 = 0.128) and total chlorophyll (r2 = 0.109) for Ambassador. This study provides new nutritional data on Se/Zn biofortified peas, important for improving agronomic biofortification of pea plants.

Solvent Precipitation SP3 (SP4) Enhances Recovery for Proteomics Sample Preparation without Magnetic Beads.

Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture protein aggregates, with subsequent washes removing contaminants. However, SP3 is dependent on effective protein immobilization onto beads, risks losses during wash steps, and exhibits losses and greater costs at higher protein inputs. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3 protein cleanup, capturing acetonitrile-induced protein aggregates by brief centrifugation rather than magnetism—with optional low-cost inert glass beads to simplify handling. SP4 recovered equivalent or greater protein yields for 1–5000 μg preparations and improved reproducibility (median protein R2 0.99 (SP4) vs 0.97 (SP3)). Deep proteome profiling revealed that SP4 yielded a greater recovery of low-solubility and transmembrane proteins than SP3, benefits to aggregating protein using 80 vs 50% organic solvent, and equivalent recovery by SP4 and S-Trap. SP4 was verified in three other labs across eight sample types and five lysis buffers—all confirming equivalent or improved proteome characterization vs SP3. With near-identical recovery, this work further illustrates protein precipitation as the primary mechanism of SP3 protein cleanup and identifies that magnetic capture risks losses, especially at higher protein concentrations and among more hydrophobic proteins. SP4 offers a minimalistic approach to protein cleanup that provides cost-effective input scalability, the option to omit beads entirely, and suggests important considerations for SP3 applications—all while retaining the speed and compatibility of SP3.

Deer antler based active ingredients have protective effects on LPS/d-GalN-induced acute liver injury in mice through MAPK and NF-κB signalling pathways

Context Deer antler based active ingredients are known to have certain anti-inflammatory and antioxidant activities. However, its potential hepatoprotective effect remains unclear. Objective This article reports the hepatoprotective effect of protein components in deer antler bases (R1) on lipopolysaccharide/d-galactosamine (LPS/d-GalN)-induced acute liver injury (ALI) in mice, and explores its possible mechanism. Materials and methods The four separated and purified protein components of deer antler bases were screened and verified by the RAW264.7 cell inflammation model. In the in vivo experiment of LPS/d-GalN-induced ALI in mice, ALT, AST, SOD, CAT, GSH and MDA were detected. The liver histopathology was analysed, the COX-2 and iNOS proteins were analysed by immunohistochemistry, and 4-HNE was analysed by immunofluorescence staining. In addition, the effects on the MAPK pathway and NF-κB/IκB-α pathway in liver proteins were explored. Results With isolated RA protein fraction pre-treated RAW264.7 cells, NO production decreased by 35.3% compared with the model group. The experimental results of ALI in mice induced by LPS/d-GalN show that R1 protein components can protect mice from ALI through anti-inflammatory and anti-oxidative stress effects and reduce liver pathological damage in mice. The results also indicate that the R1 protein component may protect the liver by inhibiting the activation of the MAPK pathway and the NF-κB/IκB-α pathway induced by LPS/d-GalN. Conclusions The separated and purified R1 protein component of deer antler base has a good protective effect on LPS/d-GalN-induced liver injury, and may become a potential material for protecting against liver injury.

The microRNA-202 as a Diagnostic Biomarker and a Potential Tumor Suppressor.

MicroRNA-202 (miR-202) is a member of the highly conserved let-7 family that was discovered in Caenorhabditis elegans and recently reported to be involved in cell differentiation and tumor biology. In humans, miR-202 was initially identified in the testis where it was suggested to play a role in spermatogenesis. Subsequent research showed that miR-202 is one of the micro-RNAs that are dysregulated in different types of cancer. During the last decade, a large number of investigations has fortified a role for miR-202 in cancer. However, its functions can be double-edged, depending on context they may be tumor suppressive or oncogenic. In this review, we highlight miR-202 as a potential diagnostic biomarker and as a suppressor of tumorigenesis and metastasis in several types of tumors. We link miR-202 expression levels in tumor types to its involved upstream and downstream signaling molecules and highlight its potential roles in carcinogenesis. Three well-known upstream long non-coding-RNAs (lncRNAs); MALAT1, NORAD, and NEAT1 target miR-202 and inhibit its tumor suppressive function thus fueling cancer progression. Studies on the downstream targets of miR-202 revealed PTEN, AKT, and various oncogenes such as metadherin (MTDH), MYCN, Forkhead box protein R2 (FOXR2) and Kirsten rat sarcoma virus (KRAS). Interestingly, an upregulated level of miR-202 was shown by most of the studies that estimated its expression level in blood or serum of cancer patients, especially in breast cancer. Reduced expression levels of miR-202 in tumor tissues were found to be associated with progression of different types of cancer. It seems likely that miR-202 is embedded in a complex regulatory network related to the nature and the sensitivity of the tumor type and therapeutic (pre)treatments. Its variable roles in tumorigenesis are mediated in part thought its oncogene effectors. However, the currently available data suggest that the involved signaling pathways determine the anti- or pro-tumorigenic outcomes of miR-202’s dysregulation and its value as a diagnostic biomarker.

Apolipoprotein (a)/Lipoprotein(a)-Induced Oxidative-Inflammatory α7-nAChR/p38 MAPK/IL-6/RhoA-GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation-Associated Development of Coronary Artery Spasm.

Objective Apolipoprotein (a)/lipoprotein(a) (Lp(a)), a major carrier of oxidized phospholipids, and α7-nicotinic acetylcholine receptor (α7-nAChR) may play an important role in the development of coronary artery spasm (CAS). In CAS, the association between Lp(a) and the α7-nAChR-modulated inflammatory macrophage polarization and activation and smooth muscle cell dysfunction remains unknown. Methods We investigated the relevance of Lp(a)/α7-nAChR signaling in patient monocyte-derived macrophages and human coronary artery smooth muscle cells (HCASMCs) using expression profile correlation analyses, fluorescence-assisted cell sorting flow cytometry, immunoblotting, quantitative real-time polymerase chain reaction, and clinicopathological analyses. Results There are increased serum Lp(a) levels (3.98-fold, p = 0.011) and macrophage population (3.30-fold, p = 0.013) in patients with CAS compared with patients without CAS. Serum Lp(a) level was positively correlated with high-sensitivity C-reactive protein (r2 = 0.48, p < 0.01), IL-6 (r2 = 0.38, p = 0.03), and α7-nAChR (r2 = 0.45, p < 0.01) in patients with CAS, but not in patients without CAS. Compared with untreated or low-density lipoprotein- (LDL-) treated macrophages, Lp(a)-treated macrophages exhibited markedly enhanced α7-nAChR mRNA expression (p < 0.01) and activity (p < 0.01), in vitro and ex vivo. Lp(a) but not LDL preferentially induced CD80+ macrophage (M1) polarization and reduced the inducible nitric oxide synthase expression and the subsequent NO production. While shRNA-mediated loss of α7-nAChR function reduced the Lp(a)-induced CD80+ macrophage pool, both shRNA and anti-IL-6 receptor tocilizumab suppressed Lp(a)-upregulated α7-nAChR, p-p38 MAPK, IL-6, and RhoA-GTP protein expression levels in cultures of patient monocyte-derived macrophages and HCASMCs. Conclusions Elevated Lp(a) levels upregulate α7-nAChR/IL-6/p38 MAPK signaling in macrophages of CAS patients and HCASMC, suggesting that Lp(a)-triggered inflammation mediates CAS through α7-nAChR/p38 MAPK/IL-6/RhoA-GTP signaling induction, macrophage M1 polarization, and HCASMC activation.

Rapid screening of unground cocoa beans based on their content of bioactive compounds by NIR spectroscopy

Near Infrared (NIR) spectroscopy was evaluated as a fast and easy method for identifying the most interesting cocoa genotypes according to chemical composition, including bioactive compound contents, in more than 80 samples of cocoa beans from the Mexican Germplasm Bank, which were harvested over three years. Clear differences in chemical composition were detected in fermented and dried samples among genotypes. The contents in fat, protein, total sugars, total phenols, phenolic compounds and theobromine were determined for both whole cocoa beans and ground cotyledon samples; and calibration models were developed from the spectra of intact beans, nibs and ground cotyledons. In general, the calibration models obtained for cotyledon composition from the spectra of cocoa nibs and ground beans were better than those obtained from the spectra of intact beans. Fat content showed better calibration statistic values from the spectra of nibs and ground cotyledon (r2 = 0.70). Bioactive compounds, such as theobromine (r2 = 0.77), total sugars (r2 = 0.74), total phenols (r2 = 0.66) and derivatives of epicatechin (r2 = 0.88), together with fat (r2 = 0.70), protein (r2 = 0.64) and husk content (r2 = 0.82), were well-predicted using NIR spectroscopy in intact beans, cocoa nibs and/or ground cotyledon. The potential of NIRS technology was confirmed to support germplasm banks and breeding programs for the rapid identification of interesting genotypes based on their contents in bioactive compounds.

Evaluation of protein descriptors in computer-aided rational protein engineering tasks and its application in property prediction in SARS-CoV-2 spike glycoprotein

The importance of protein engineering in the research and development of biopharmaceuticals and biomaterials has increased. Machine learning in computer-aided protein engineering can markedly reduce the experimental effort in identifying optimal sequences that satisfy the desired properties from a large number of possible protein sequences. To develop general protein descriptors for computer-aided protein engineering tasks, we devised new protein descriptors, one sequence-based descriptor (PCgrades), and three structure-based descriptors (PCspairs, 3D-SPIEs_5.4 Å, and 3D-SPIEs_8Å). While the PCgrades and PCspairs include general and statistical information in physicochemical properties in single and pairwise amino acids respectively, the 3D-SPIEs include specific and quantum–mechanical information with parameterized quantum mechanical calculations (FMO2-DFTB3/D/PCM). To evaluate the protein descriptors, we made prediction models with the new descriptors and previously developed descriptors for diverse protein datasets including protein expression and binding affinity change in SARS-CoV-2 spike glycoprotein. As a result, the newly devised descriptors showed a good performance in diverse datasets, in which the PCspairs showed the best performance (R2=0.783 for protein expression and R2=0.711 for binding affinity). As a result, the newly devised descriptors showed a good performance in diverse datasets, in which the PCspairs showed the best performance. Similar approaches with those descriptors would be promising and useful if the prediction models are trained with sufficient quantitative experimental data from high-throughput assays for industrial enzymes or protein drugs.

Structural and Biochemical Investigation of Class I Ribonucleotide Reductase from the Hyperthermophile Aquifex aeolicus.

Ribonucleotide reductase (RNR) is an essential enzyme with a complex mechanism of allosteric regulation found in nearly all living organisms. Class I RNRs are composed of two proteins, a large α-subunit (R1) and a smaller β-subunit (R2) that exist as homodimers, that combine to form an active heterotetramer. Aquifex aeolicus is a hyperthermophilic bacterium with an unusual RNR encoding a 346-residue intein in the DNA sequence encoding its R2 subunit. We present the first structures of the A. aeolicus R1 and R2 (AaR1 and AaR2, respectively) proteins as well as the biophysical and biochemical characterization of active and inactive A. aeolicus RNR. While the active oligomeric state and activity regulation of A. aeolicus RNR are similar to those of other characterized RNRs, the X-ray crystal structures also reveal distinct features and adaptations. Specifically, AaR1 contains a β-hairpin hook structure at the dimer interface, which has an interesting π-stacking interaction absent in other members of the NrdAh subclass, and its ATP cone houses two ATP molecules. We determined structures of two AaR2 proteins: one purified from a construct lacking the intein (AaR2) and a second purified from a construct including the intein sequence (AaR2_genomic). These structures in the context of metal content analysis and activity data indicate that AaR2_genomic displays much higher iron occupancy and activity compared to AaR2, suggesting that the intein is important for facilitating complete iron incorporation, particularly in the Fe2 site of the mature R2 protein, which may be important for the survival of A. aeolicus in low-oxygen environments.

Serum erythroferrone levels during the first month of life in premature infants.

To examine erythroferrone (ERFE)-hepcidin iron regulation in premature infants under intensive care at risk of iron metabolic disorders. A retrospective cohort recruited 31 infants with a birth weight of <1500 g hospitalized in a tertiary center. Their hematological status was measured at birth and 2 and 4 weeks of life. ERFE was positively correlated with the reticulocyte hemoglobin content at 2 (r2 = 0.2374) and 4 weeks (r2 = 0.6005). An assumed negative correlation between ERFE and hepcidin was not determined during the neonatal period. Hepcidin was positively correlated with the leukocyte count (r2 = 0.3089) and ferritin (r2 = 0.7476) at birth and C-reactive protein (r2 = 0.3591) at 2 weeks and negatively correlated with the reticulocyte count (r2 = 0.2887) at 4 weeks. The vulnerability of the ERFE-hepcidin pathway within 4 weeks may contribute to iron imbalance in premature infants.

Interferon-γ Induces Distinct mRNA and Protein Profiles in Acute and Chronic Myeloid Leukemia

Leukemia cells promote tumor survival and progression by avoiding host immune surveillance. A key pathway involves indoleamine 2,3-dioxygenase-1 (IDO1), an interferon (IFN)-γ-inducible molecule mediating feedback inhibition and hindering anti-tumor immunity. We have previously shown thattreatment of primary acute myeloid leukemia (AML) cells with IFN-γ in vitro induces functional IDO1 in 50% of unselected cases (‘IFN-γ responders‘), an effect which was abrogated by STAT3 inhibitors. With a median follow-up of 8 years, these IFN-γ responders experienced a shorter event-free survival and overall survival. The current study was undertaken to dissect the intracellular signaling pathways that are activated in leukemia cells in response to IFN-γ.We employed an alpha version of the Vantage 3D™ DNA:RNA:Protein Heme Assay (NanoString Technologies Inc., Seattle, USA) to profile DNA mutations, RNA, protein and phosphoprotein simultaneously. The assay profiles 180 mRNA species involved in hematology/oncology pathways, 38 total and phosphorylated proteins (including JAK-STAT, Src, BTK, FLT3, PI3K-Akt, NF-kB), and 124 single nucleotide variants (SNVs) relevant to hematological malignancies (IDH, FLT3, KRAS, NRAS, DNMT3, JAK3 and others). K562 and TCC-S cells (chronic myeloid leukemia [CML] in blast crisis), KG1a cells (leukemia stem cell [LSC]-like AML) and Kasumi-1 cells [AML with t(8;21)] were stimulated with 100 ng/mL IFN-γ then harvested and lysed after 30m, 1h, 2h, 12h and 24h for processing in parallel workflows (protein/mRNA and DNA SNV assays) prior to overnight hybridization with NanoString reporters. Pooled RNA/DNA/protein samples were analyzed using the nCounter® FLEX platform and the nSolver® software (alpha version 4.0). CCRF-CEM and HUT78 cell lines, with known DNA variants and expression of protein targets represented in the 3D Heme panel, were used as controls.K562, TCC-S and Kasumi-1 cells, but not KG1a cells, up-regulated IDO1 mRNA after IFN-γ challenge. IFN-γ-sensitive CML cells (K562 and TCC-S) expressed high levels of mRNA transcripts for JAK-STAT intermediates and genes implicated in apoptosis, DNA damage and cytokine signaling (Fig. 1). IFN-γ-sensitive t(8;21) AML cells displayed a distinct transcriptomic profile with an up-regulated subset of IFN-inducible genes, including IRF1 , STAT1, BCL3, SOCS1 , PD-L1 , ISG15 , OSM , HLA class I and genes involved in antigen processing (CIITA ). In contrast, the transcriptome of IFN-γ-insensitive LSC-like KG1a cells consisted of high levels of genes implicated in epigenetics and transcription, NF-κB, MYC, B-cell receptor and T-cell receptor signaling, and low expression levels of genes mediating JAK-STAT signaling, apoptosis, DNA damage and cytokine signaling. Overall phosphorylation of intracellular proteins was largely unchanged after IFN-γ stimulation of KG1a cells. In contrast, IFN-γ activation of Kasumi-1 AML cells was associated with increased phosphorylation of Akt(S473), STAT3(Y705) and proline-rich Akt substrate of 40kD (PRAS40) (T246). Similar phosphorylation changes were apparent in TCC-S CML cells, although with lower magnitude. Interestingly, HOXA9 transcription factor, which is commonly dysregulated in acute leukemias, was highly expressed, both as mRNA and protein (r2=0.912; p<0.001), in LSC-like KG1a cells, but not in t(8;21) AML and CML cell lines. SNV analysis identified all expected gene mutations in the control cell lines and TP53(R248Q) in Kasumi-1 cells.The use of artificial neural network algorithms and a public collection of AML transcriptomic data (641 patients; E-MTAB-3444) allowed us to identify enriched functional-related gene groups (‘immune response’, ‘antigen processing and presentation’, ‘MAPK signaling pathway’; DAVID v6.8), the expression of which was predicted by the 3 most up-regulated transcripts in our IFN-γ-sensitive leukemia cells (IRF1, STAT1 and BCL3 ).In conclusion, the combined analysis of SNVs, mRNA and protein expression on the nCounter® platform is a powerful tool to characterize the molecular landscape of leukemias. Our analysis of IFN-γ-stimulated leukemia cells has identified molecular pathways that could be targeted in patients with ‘inflamed’ tumors, in which microenvironmental production of IFN-γ is heightened due to ongoing T-cell responses.Grant support: Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494). [Display omitted] DisclosuresMeredith:NanoString Technologies: Employment. Ross:NanoString Technologies: Employment. Demirkan:NanoString Technologies: Employment. Rutella:NanoString Technologies, Seattle, USA: Research Funding.